1. Field of the Invention
The present invention relates to a device and a method for the quick and easy assay of a liquid sample, particularly body liquids, such as whole blood, blood serum, blood plasma, urea and cerebrospinal liquid.
2. Description of the Related Art
For the assay of a particular component in a liquid sample, an enzymatic analysis is widely used since it can be carried out under mild conditions. In clinical tests, the body liquid is analyzed with a reagent solution containing an oxidase which oxidizes a particular component, a pigment precursor and, optionally, a peroxidase. In such an enzymatic analysis, hydrogen peroxide, which is generated through oxidation of the particular component with the oxidase, directly oxidizes the oxidizable pigment precursor or oxidation coupling with the pigment precursor in the presence of the peroxidase to generate a pigment. Then, the generated pigment is colorimetrically measured by, for example, absorptiometry, fluorophotometry and emission spectroscopy, and in turn, the amount or concentration of particular component is indirectly measured.
Examples of the oxidases useful in the clinical tests are glucose oxidase, uricase, cholesterol oxidase, glycerol-triphosphate oxidase, cholin oxidase, acyl-CoA oxidase, sarcosine oxidase, various amino acid oxidases, bilirubin oxidase, lactate oxidase, lactose oxidase, pyruvate oxidase, galactose oxidase, glycerol oxidase and the like.
As the pigment precursor, oxidizable pigment precursors such as a so-called Trinder's reagent (cf. Ann. Clin. Biochem., 6, 24 (1960)), o-anisidine, benzidine, o-tolidine and tetramethylbenzidine are well known.
One of the advantages of the enzymatic analysis resides in that, if the component to be analyzed is changed, the same color developing system can be used by changing the kind of oxidase. Therefore, the application of the enzymatic analysis to various analytical items has been investigated.
Recently, in order to quickly and conveniently carry out the assay, a reagent system fixed on a solid support has been advantageously used in place of the conventional reagent solution.
For example, U.S. Pat. No. 3,630,957 and Japanese Patent Publication No. 33800/1974 disclose a water-resistant test film which comprises a plastic film and a polymer layer in which an assay system comprising the above oxidase and hydrogen peroxide is dispersed. In the assay with such water-resistant test film, since the color of the generated pigment is measured from the side on which the sample, such as the whole blood or the blood serum, is applied, the sample should be wiped out with a piece of absorbent cotton and the like after contacting the sample to the reagent layer for a certain period of time. Further, since the color is developed in the presence of a sufficient amount of oxygen after wiping out of the sample, the test film should be kept standing in the air for a certain period of time.
U.S. Pat. No. 3,992,158 and Japanese Patent Publication No. 21677/1978 disclose a multi-layer test film comprising a liquid-impermeable transparent support, a reagent layer and a spreading layer. When the liquid sample is applied at a point of the spreading layer, it spreads over the spreading layer and then migrates into the reagent layer. The color of the pigment which is generated through the reaction between the specific component and the reagent is measured through the transparent support. Therefore, it is not necessary to remove the applied sample. However, since the reagent layer is present between the support and the spreading layer, it is difficult for the air to reach the reagent layer through the spreading layer. Particularly when the reagent layer contains the oxidase, the reaction does not proceed sufficiently due to shortage of oxygen.
To overcome this drawback, EP-A-137 521 and Japanese Patent Kokai Publication No. 82859/1985 discloses a multi-layer integral element for chemical analysis, which comprises an oxygen-permeable protein-impermeable light shielding layer between the reagent layer and the spreading layer to improve the contact between the reagent layer and the air. However, since the reagent layer never contacts the air on the support side, oxygen is not sufficiently supplied to the reagent layer.
German Patent No. 3 510 992 and Japanese Patent Kokai Publication No. 205364/1985 disclose an assay device having a porous hydrophobic oxygen-supplying layer between the reagent layer and the support. Such a device can significantly increase the amount of oxygen supplied to the reagent layer containing the oxidase. But, since the oxygen-supplying layer has a certain thickness to retain a sufficient amount of oxygen therein, the device has no light-transparency on the support side, so that the color of the generated pigment should be measured from the sample-supplied side of the device.